The long term objectives are to delineate the action mechanisms of human DNA polymerase i (Poll) on undamaged and damaged DNAs. The specific aims of this project are to examine the role of Hoogsteen base pairing in the replication of undamaged and damaged DNAs by Poll, and the proposed studies will use a combined biochemical and structural approach, hi Aim 1, biochemical studies will be done with base analogs of adenine and guanine to analyze the contributions of different hydrogen bond acceptors/donors in template purines to their Hoogsteen pairing with the incoming pyrimidine nucleotide (nt). Aim 2 will test the hypothesis that Hoogsteen base pairing enables Poll to incorporate nts opposite DNA lesions that severely impinge upon the minor groove of the template base or which impair its Watson-Crick base pairing ability. The proficiency of Poll for incorporating nts opposite such lesions and to extend there from will be determined by steady-state kinetic methods. Aim 3 will determine the structural bases of Hoogsteen base pairing in Poll's active site by analyzing the effects that mutational changes in the residues of the fingers and palm domains, that are seen to interact with the templating nt or with the incoming nt in Poll ternary structure, have on the efficiency and fidelity of nt incorporation, hi Aim 4, the X-ray structures of ternary complexes of Poll with undamaged templates G, T, and C, in the presence of different incoming nts will be determined, and in Aim 5, the ternary structures of Poll with DNA containing lesions at the templating site that severely impinge upon the minor groove, or which affect Watson-Crick base pairing, and others, in the presence of different incoming nts will be determined. The results will be highly relevant for cancer biology, as the manner in which DNA lesions are bypassed during replication has a major impact on genome stability; and, in fact, the inactivation of Polr in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum.